WO2010111883A1 - 药敏检验方法及其装置 - Google Patents

药敏检验方法及其装置 Download PDF

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WO2010111883A1
WO2010111883A1 PCT/CN2010/000064 CN2010000064W WO2010111883A1 WO 2010111883 A1 WO2010111883 A1 WO 2010111883A1 CN 2010000064 W CN2010000064 W CN 2010000064W WO 2010111883 A1 WO2010111883 A1 WO 2010111883A1
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Prior art keywords
drug
test
susceptibility
culture solution
bacteria
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PCT/CN2010/000064
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English (en)
French (fr)
Inventor
彭钧
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湖南省天骑医学新技术有限公司
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Priority to RU2011125572/10A priority Critical patent/RU2011125572A/ru
Priority to JP2011528176A priority patent/JP2012503975A/ja
Priority to EP10757986A priority patent/EP2415874A4/en
Publication of WO2010111883A1 publication Critical patent/WO2010111883A1/zh
Priority to US13/031,597 priority patent/US20110143390A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements

Definitions

  • the invention relates to a bacteria, a cell drug sensitivity test method and a drug sensitivity test device.
  • Drug susceptibility testing is an important test for the detection of bacterial or cellular resistance.
  • the existing drug sensitivity test method generally uses two kinds of mediums: a liquid medium and a solid medium.
  • the test method generally includes a paper test method and a concentration test method, and the paper test method uses a solid medium.
  • the concentration test uses a liquid medium.
  • the paper test method is to determine the sensitivity of Mycobacterium tuberculosis to drugs by observing the drug inhibition zone formed by the drug paper attached to the surface of the solid medium; the concentration test method is to observe different drugs and different drug concentration test containers by observation.
  • the growth of bacteria or cells to determine the sensitivity of the bacteria or cells to the drug.
  • the entire drug sensitivity test process can be generally divided into three stages: concentrated bacteria or cells, proliferation or separation culture and drug sensitivity test.
  • the susceptibility test of Mycobacterium tuberculosis using the existing technology generally has a paper test method and a concentration test method, and the whole test process can be generally divided into three stages: concentrated Mycobacterium tuberculosis, proliferation or isolation culture and drug susceptibility test. .
  • the steps of accumulating the Mycobacterium tuberculosis stage are as follows: 1 using a pipette to take the specimen provided by the patient into the test container; 2 adding the digestive juice to the test container, digesting the sample mucus, and making the mucus in the sample The bacteria enclosed in the impurities are fully exposed; 3 centrifugation, the supernatant is discarded, and the tuberculosis specimen is obtained. After obtaining the Mycobacterium tuberculosis specimen, proliferation or isolation culture is carried out.
  • the steps of proliferating or isolating the culture and culture stage are as follows: (4) taking a small portion of the already concentrated tubercle bacillus specimen to prepare a concentrated liquid solution; 5 inoculating the concentrated liquid solution on a solid medium such as Roche medium for proliferation or isolation culture, generally It needs to be cultured in a 37-inch incubator for about 1 month, and the individual bacteria are cultured into bacterial colonies; after obtaining the bacterial bacteria, they enter the drug sensitivity test stage.
  • Drug sensitivity test The steps of the section are as follows: 6 taking a small amount of bacterial colonies, generally taking one to three bacterial colonies, dissolving and dispersing into a concentrated liquid; 7 inoculating the concentrated liquid on a surface of a solid medium such as an agar medium; 8 on the surface of the solid medium paste the drug paper; 9 placed in a 37 ° C incubator for about 1 month, observe the drug inhibition zone, determine the sensitivity of Mycobacterium tuberculosis to drugs.
  • the advantages of the existing paper inspection method are that the operation is convenient, the equipment is simple, and the investment is small; however, it has the following disadvantages: (1) The time required for the entire inspection is too long.
  • concentration test method In the existing concentration test method, there are two methods.
  • One concentration test method is to use a solid medium in the proliferation culture stage, and the steps of accumulating Mycobacterium tuberculosis and the two stages of proliferation or isolation culture are compared with the existing paper test. The method is the same, but the steps in the drug susceptibility test phase are different from the existing paper test method.
  • the bacterial colonies obtained by the proliferation or separation culture culture are taken from the solid medium, and generally one or several bacterial colonies are taken.
  • the advantage of this test method is that the equipment is simple and the investment is small; however, it has the following disadvantages: (1) The time required is long. The method and procedure for proliferating or isolating the culture and culture stage are the same as those of the existing paper test method. Therefore, it takes about one month to proliferate or separate the time required for the culture and culture stage; (2) troublesome operation Insecure and prone to erroneous test results.
  • Another concentration test is to use a liquid medium in the proliferation or isolation culture stage. This test method is generally tested by special instruments, such as BACTEC 9000MB and BACTEC MGIT 960 automatic rapid mycobacterial culture identification drug system introduced by BD. The advantages are simple operation, short inspection time and average detection time of 14 4 days, the fastest 10 days or so.
  • the object of the present invention is to provide a bacteria, cell susceptibility test method and drug susceptibility test device which have a short test process time; another object is low investment and low use cost; and another object is safe and convenient to use.
  • the method for testing bacterial and cellular susceptibility of the present invention comprises:
  • a liquid test specimen is added with a coagulant which can react with the colloidal raw material to form a gel, and is prepared into a gel test specimen;
  • the culture solution comprises a bacterial cell growth indicator.
  • the liquid test specimen is placed in an incubator for proliferation for 2 to 4 days.
  • the culture solution includes a bacteriostatic agent.
  • the ratio between the nutrient substance and the colloidal material in the culture solution is: 0.8 g - 2 g of the colloidal material per 100 ml of the nutrient substance ; and the coagulant O for every 100 ml of the nutrient substance Lg - 0.2g.
  • the above bacteria, cell susceptibility test method, the ratio between the nutrient, the colloidal material, and the bacterial cell growth indicator in the culture solution is: adding a colloidal material per 100 ml of the nutrient substance
  • the ratio between the nutrient substance, the colloidal material, the bacterial cell growth indicator and the bacteriostatic agent in the culture solution is: 0.8 g of colloidal material per 100 ml of nutrient substance --- 2g ; add bacterial cell growth indicator 2mg - 20mg ; add appropriate amount of bacteriostatic agent; per 100ml nutrient with coagulant O.lg - 0.2go.
  • the culture solution is dispensed and stored in a sealed quantity in a single use.
  • the susceptibility testing device for the above-mentioned bacteria and cell susceptibility testing method comprises a box body having a peripheral wall and a bottom closed structure and an open upper end structure, and a coagulant is placed in the inner cavity of the box body.
  • the above drug sensitivity testing device is provided with a coagulating layer at the bottom of the inner cavity of the box, and the coagulating layer is composed of a sustained release body and a coagulant attached to the sustained release body.
  • the drug sensitivity testing device is provided with a coagulating layer at the bottom of the inner cavity of the box, and the coagulating layer is a structure in which the adhesive and the coagulant are mixed and applied to the bottom of the box body.
  • the drug sensitivity testing device described above is provided with a scale at the bottom of the casing.
  • the drug sensitivity testing device described above is provided with a lid attached thereto at the open end of the casing.
  • the nutrient in the culture solution comprises a nutrient component and a buffering agent, and the function is to provide nutrition for the growth of bacteria or cells and control the pH of the culture solution.
  • Different nutrient formulations are available for different bacteria or cells.
  • tubercle bacilli can be selected from 7H9 formula
  • common bacteria or fungi can be selected from nutrient broth formula
  • tumor cells can be selected from cell culture medium.
  • the colloidal material in the culture solution functions to dissolve the colloidal material before the coagulant is added to the culture solution, so that the culture solution is in a liquid state, and after the coagulant is added to the culture solution, the colloidal material reacts with the coagulant to make
  • the culture solution forms a stable gel within a certain period of time, and may be selected from 0.5 to 1.5% sodium alginate or 0.5 to 2.0% pectin, 0.5 to 1.5% carrageenan or the like.
  • the function of the bacterial cell growth indicator in the culture solution is that the bacteria or cells will decompose the MTT during the growth and reproduction to form a blue dot-like chromophore, thereby forming a blue macroscopic particle for each bacteria or cell in a short time.
  • the growth state of cells or bacteria can be determined according to the nature and quantity of the naked eye or the low magnification microscope. 3-(4, 5-dimethylthiazole-2)-2, 5-diphenyl can be used.
  • 4-azole bromide (trade name is thiazolyl blue, English name
  • the bacteriostatic agent added to the culture solution is mainly used for the susceptibility test of Mycobacterium tuberculosis, and its action is to inhibit the growth of other bacteria other than acid-fast bacilli.
  • the coagulant used in the bacterial and cell susceptibility test method of the present invention acts to react the coagulant with the colloidal material in the culture solution to form a stable gel in a certain period of time.
  • Calcium or magnesium ions, borax, potassium citrate, etc. may be used.
  • the drug susceptibility testing device comprises a box body having a peripheral wall and a bottom closed structure and an open upper end structure, a coagulant is placed in the box body, and a colloidal material is added to the culture liquid.
  • the liquid test specimen is made of the culture liquid. Therefore, there is a colloidal material in the liquid test specimen.
  • the liquid test specimen is liquid before being poured into the drug sensitivity test device. After pouring the drug sensitivity test device, the test specimen is inspected.
  • the colloidal raw material reacts with the coagulant in the drug sensitivity test device, and can form a stable gel form at normal temperature to form a gel test specimen, so that the drug paper sheet can be attached to the surface of the gel test specimen. Therefore, drug paper can be used to test the sensitivity of bacteria or cells to drugs.
  • the reason for the short time of the whole testing process is as follows: 1. Fully utilizing the bacteria or cells to be tested in the specimen provided by the patient. Most of the bacteria or cells in the specimens provided by the patients are concentrated for the production of test specimens, and the test specimens are all used for the susceptibility test. Therefore, the bacteria or cells to be tested in the specimens provided by the patients are fully obtained. Use. 2 The proliferation or isolation culture stage can be omitted or the proliferation or isolation culture time can be reduced. The drug sensitivity test is performed on the general bacteria or cells, and the six drug paper test can satisfy the requirements. Therefore, the area of the cavity of the drug sensitivity test device can be designed to simultaneously perform the drug sensitivity test of six drug paper sheets.
  • drug susceptibility testing is the detection of bacterial or cell resistance
  • drug susceptibility testing Previously, patients were diagnosed.
  • the bacteria and cell susceptibility test method of the present invention makes full use of the bacteria or cells in the specimen provided by the sample, the unit area of the inner cavity of the drug susceptibility test device is tested when performing the susceptibility test.
  • the number of bacteria or cells is large enough to meet the number of bacteria or cells required for the susceptibility test, and it is no longer necessary to increase the number of bacteria or cells by proliferation culture.
  • the specimen provided by the patient contains less bacteria to be tested, and the prepared liquid test specimen should be placed in an incubator for proliferation culture, and then the liquid test specimen is poured into the medicine (in the test device) Susceptibility test. Since the proliferation culture of the present invention is carried out in a liquid medium, relatively, the proliferation rate of the bacteria in the liquid medium is faster than that on the solid medium, and the patient is fully utilized. The sample to be inspected is fine, so that the initial amount before the proliferation culture is large, and therefore, the time of proliferation culture is very short.
  • the bacteria circle which takes less time than the existing paper test method, saves the entire test time. Together with the display of the bacterial cell growth indicator, it can better show the growth of bacteria or cells. And the drug inhibition zone is more convenient for observation and detection, and the test time can be further reduced.
  • the susceptibility test of Mycobacterium tuberculosis can be carried out by using the bacteria and cell susceptibility test method of the present invention, and the proliferation culture stage can be omitted, even if proliferation is required.
  • Cultivate also It takes only 2-4 days, and the drug sensitivity test phase only takes 5-7 days, and the entire inspection process is less than 14 days. In summary, the present invention achieves the purpose of short inspection time.
  • the reason why the bacteria and cell susceptibility test method of the present invention have low investment and low use cost is that the equipment required for carrying out the invention is basically the same as the existing paper test method, and only a centrifugal device, a drug susceptibility test device, and the like are required.
  • the above-mentioned centrifugal device and drug susceptibility testing device are simple to manufacture and low in production cost, and therefore, the investment in the whole set of equipment is small; the implementation of the present invention requires the use of a disposable test container and reagents including nutrients, buffers, colloidal materials, indicators, and the like.
  • the reason why the bacteria and cell drug sensitivity test method of the invention is safe and convenient to use is as follows:
  • the invention can concentrate the sample to be inspected and the result accompanying reading can be carried out in a disposable closed container, does not pollute other equipment, and is uniformly disinfected after the operation is completed. And the procedures for manual operation throughout the inspection process are few and simple, thus minimizing the impact of bacteria on workers and the environment, greatly improving the biosafety of the test.
  • the culture solution for one use is filled in a sealed bottle, when it is used, it is only necessary to open the sealed bottle, mix the culture solution with the concentrated bacteria or cells, and then pour it into the drug sensitivity test device.
  • the operation is simple and convenient, and therefore, the invention can effectively realize the purpose of safe and convenient use.
  • Fig. 1 is a cross-sectional view showing the main portion of the drug susceptibility testing device of the present invention.
  • Figure 2 is a bottom plan view of Figure 1. 1, box, 2, coagulation layer, 3, lid, 4, ruler.
  • the casing (1) is a structural form in which the peripheral wall and the bottom are closed and the upper end is open, and a coagulating layer (2) is provided in the casing (1), and the coagulating layer (2) is provided by the sustained release body and the adhesion.
  • the coagulant composition in the sustained release body is provided with a scale (4) at the bottom of the casing (1), and a lid is provided at the open end of the casing (1)
  • the sustained release body in the coagulating layer (2) may be a sustained release film, and when the culture solution is poured into the cartridge (1), the sustained release film may be dissolved in the culture solution.
  • the coagulant is attached to the sustained release body; the sugar may be mixed with the coagulant to form a coagulating layer in the casing (1) by spraying.
  • the precipitate is eluted with 20 ml of the culture liquid to prepare a liquid test specimen in the form of a liquid bacterial suspension;
  • the liquid test specimen is evenly poured into the box of the drug sensitivity test device, and the colloidal material in the culture solution reacts with the coagulant in the cartridge to solidify into a stable gel within 3 hours to form a gel test mark. 7. Cover the lid and place it in a 37 ° C incubator for 5-7 days. During the growth and reproduction process, Mycobacterium tuberculosis will decompose MTT to form a blue dot-like chromophore. If the drug has inhibitory effect on Mycobacterium tuberculosis, A gel test specimen attached to a drug paper sheet cannot form a blue cluster around the specimen, thereby forming a zone of inhibition;
  • the 20 ml culture solution in this embodiment is formulated as follows: the nutrient is 20 ml of 7H9 formula, and the bacterial cell growth indicator is 3-(4,5-dimethylthiazole-2)-2, 5-diphenyltetra
  • the azole salt is 0.4 mg
  • the colloidal material is 0.5-1.5% sodium alginate 0.11 g
  • the inhibitor is 0.4 mg malachite green
  • the coagulant in this example is 0.02 g of calcium ion.
  • the formulation of the 20 ml culture solution in this embodiment may also be: the nutrient is 20 ml of the 7H9 formula, and the bacterial cell growth indicator is 3-(4,5-dimethylthiazole-2)-2, 5-diphenyl.
  • the tetrazolium bromide salt is 0.5 mg
  • the colloidal material is 0.5-1.5% sodium alginate 0.3 g
  • the inhibitor is 0.4 mg malachite green 0.8 ml
  • the coagulant in this embodiment is 0.03 g of calcium ion.
  • the formulation of the 30 ml culture solution in this embodiment may also be: the nutrient is 20 ml of the 7H9 formula, and the bacterial cell growth indicator is 3-(4,5-dimethylthiazole-2)-2, 5-diphenyl. 4mg ⁇ The tetrazolium bromide 4mg, the colloidal material 0. 4g, the inhibitor is 0. 4mg malachite green. The coagulant in this example was 0.04 g of calcium ion.
  • the precipitate is eluted with 20 ml of the culture liquid to prepare a liquid test specimen in the form of a liquid bacterial suspension; 5.
  • the liquid test specimens were placed in a 37 ° C incubator for 2-4 days, and the liquid test specimens were observed to have a pale blue color change;
  • the liquid test specimen after enrichment is evenly poured into the box of the drug sensitivity test device.
  • the colloidal material in the culture solution reacts with the coagulant in the cartridge to solidify into a stable gel within 3 hours.
  • Mycobacterium tuberculosis will decompose MTT to form a blue punctate, if the drug inhibits M. tuberculosis.
  • the formulation of the 20 ml culture solution in this embodiment may also be: the nutrient is 20 ml of the 7H9 formula, and the bacterial cell growth indicator is 3-(4,5-dimethylthiazole-2)-2, 5-diphenyl. 4mg ⁇
  • the tetrazolium bromide salt 0.5mg
  • the colloidal material is 0.5-1.5% sodium alginate 0. 3g
  • the inhibitor is 0. 4mg malachite green.
  • the coagulant in this example was 0.03 g of calcium ion.
  • the target colony after separation and culture is made into a liquid test specimen in the form of a liquid bacterial suspension using 20 ml of the culture solution;
  • the liquid test specimen is evenly poured into the box of the drug sensitivity test device.
  • the colloidal material in the culture solution reacts with the coagulant in the cartridge to solidify into a stable gel within 3 hours to form a gel test specimen.
  • the formulation of the 20 ml culture solution in this embodiment is as follows: the nutrient is 20 ml of the nutrient broth formula, and the bacterial cell growth indicator is 3-(4,5-dimethylthiazole-2)-2, 5-diphenyl. 4 ⁇
  • the tetrazolium bromide salt 4mg, the colloidal material is 0.5-1.5% sodium alginate 0. 4g; the coagulant in this example, is calcium ion 0.04g.
  • bacteria The following examples of the bacteria, cell susceptibility test method and drug susceptibility test device for tumor cell susceptibility test are given below:
  • the liquid test specimen is evenly poured into the box of the drug sensitivity test device.
  • the colloidal material in the culture solution is combined with the coagulant placed in the cartridge to solidify into a stable gel within 3 hours to form a gel test.
  • the 20 ml culture solution in this embodiment is formulated as follows: the nutrient is 20 ml of the cell culture medium formula, and the bacterial cell growth indicator is 3-(4,5-dimethylthiazole-2)-2, 5-diphenyl. 5 mg of tetrazolium bromide salt, 0.5-1.5% sodium alginate colloidal material 0. 4m go The coagulant in this example is 0.04 mg of calcium ion.

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Description

药敏检验方法及其装置 技术领域
本发明涉及一种细菌、 细胞药敏检验方法及药敏检验装置。
背景技术
药敏检验是对细菌或细胞耐药性进行检测的重要试验。 现有的药敏检验 方法， 其用的培养基一般分为液体培养基和固体培养基两种， 其检验方法一 般有纸片检验法和浓度检验法， 纸片检验法采用的是固体培养基， 浓度检验 法采用的是液体培养基。 纸片检验法是通过观察由于贴在固体培养基表面上 药物纸片形成的药物抑菌圈来确定结核杆菌对药物的敏感性； 浓度检验法是 通过观察检测不同药物和不同药物浓度试验容器中细菌或细胞的生长情况来 确定细菌或细胞对药物的敏感性。 不管是纸片检验法还是浓度检验法， 其整 个药敏检验过程一般可分为浓集细菌或细胞、 增殖或分离培养和药敏检验三 个阶段。 例如利用现有的技术对结核杆菌进行药敏试验， 一般有纸片检验法 和浓度检验法， 其整个检验过程一般都可以分为浓集结核杆菌、 增殖或分离 培养和药敏检验三个阶段。 现有的纸片检验法， 浓集结核杆菌阶段的步骤为: ①用吸管吸取患者提供的标本置于试验容器中； ②向试验容器中加入消化液， 对标本粘液进行消化， 使标本中粘液和杂质中包裹的细菌充分暴露； ③离心 分离， 倒掉上清液， 获得结核杆菌标本。 在获得结核杆菌标本后再进行增殖 或分离培养培养。 增殖或分离培养培养阶段的步骤为： ④取已经浓集的结核 杆菌标本少部分制成浓菌液； ⑤将浓菌液接种于固体培养基例如罗氏培养基 上进行增殖或分离培养培养， 一般需在 37Ό培养箱中培养 1月左右， 把单个 细菌培养成细菌菌落； 在获得细菌菌落后再进入药敏检验阶段。 药敏检验阶 段的步骤为： ⑥取少量的细菌菌落， 一般是取 1一一 3个细菌菌落， 将^ ί溶解 分散成浓菌液； ⑦将浓菌液接种于固体培养基例如琼脂培养基表面上; ⑧在 . 固体培养基表面上贴上药物纸片； ⑨放入 37°C培养箱中培养 1个月左右， 观 察药物抑菌圈， 确定结核杆菌对药物的敏感性。 现有的纸片检验法的优点是 操作方便、 设备简单， 投资小； 但其存在以下缺点： （1)整个检验所需的时间 太长。 增殖或分离培养培养阶段需要 1个月左右， 药敏检验阶段大约需要 1 个月左右， 整个检验过程大约需要 2个月左右。 如果在药敏检验得到结果后 再使用药物治疗， 就会严重影响治疗时机。 而临床上在没有药敏检验的情况 下使用药物治疗往往会造结核杆菌耐药的严重后果。 （2)不安全。 整个检验需 要两次人工接种， 由于结核杆菌具有传染性， 这样对操作人员形成安全隐患， 对环境形成污染隐患。 ·
现有的浓度检验法， 有两种方法， 一种浓度检验法是在增殖培养阶段采 用固体培养基， 其浓集结核杆菌和增殖或分离培养培养二个阶段的步骤与现 有的纸片检验法相同， 但药敏检验阶段的步骤与现有的纸片检验法不同， 具 体为： 从固体培养基上取增殖或分离培养培养获得的细菌菌落， 一般是取 1 或数个细菌菌落， 一一将其溶解分散成菌悬液一一^菌悬液倒入按不同的药 物和不同的浓度配制成的多个试验容器中， 制成对比试验标本→→观察检测 试验容器中细菌生长情况， 确定结核杆菌对药物的敏感性。 这种检验方法的 优点是设备简单， 投资小； 但其存在以下缺点： （1)所需的时间长。 增殖或分 离培养培养阶段的方法和步骤与现有的纸片检验法的方法和步骤相同， 因此， . 仅增殖或分离培养培养阶段所需的时间就需要 1个月左右； （2)操作麻烦、 不 安全且容易造成错误的检验结果。 由于需要配制多个试验容器， 配制过程很 麻烦且不安全， 对操作人员形成安全隐患， 对环境形成污染隐患； 在配制多 个试验容器过程中比较容易被其他细菌污染， 这样就可能造成错误的检验结 果。 另一种浓度检验法是在增殖或分离培养培养阶段采用液体培养基。 这种 检验方法一般采用专用仪器检验，例如 BD公司推出的 BACTEC 9000MB及 BACTEC MGIT 960全自动快速分枝杆菌培养鉴定药敏系统， 其优点是操作简单， 检验 过程时间短， 平均检出时间为 14. 4天，最快 10天左右。 但其存在的主要缺点 是仪器设备的价位高， 投资大； 使用成本高， 一是仪器设备的使用成本高， 二是需要多个对比试验标本， 试剂的成本较高， 其费用较大， 一般医院难以 承受， 难以推广普及。 .
对体外细胞进行药敏试验的方法较多， 常用的方法有 MTT比色法、 放射 线同位素掺入法等， 但其都存在操作麻烦、 容易造成错误的检验结果等缺点。 原因是需要配制不同药物和不同浓度的多个试验容器， 并且要向每个试验容 器中加细胞悬液， 配制和加细胞悬液的过程很麻烦， 在配 $ij多个试验容器过 ' 程中比较容易被污染， 这样就可能造成错误的检验结果。 · 从上面详细分析结核杆菌药敏检验中可以看出， 现有的细菌、 细胞药敏 检验方法尤其是结核杆菌检验方法存在以下缺点： （1 ) 整个检验过程所需的 时间长。 （2 ) 操作麻烦。 （3 ) 不安全。 （4) 容易出现误检。 （5 ) 投资大， 难以推广普及。
发明内容
• 本发明的目的是提供一种检验过程时间短的细菌、 细胞药敏检验方法及 药敏检验装置； 另一个目的是投资少、 使用成本低； 再一个目的是使用安全 方便。 - 为实现上述目的， 本发明细菌、 细胞药敏检验方法， 包括：
( 1 ) 浓集待检细菌标本或细胞标本； (2 ) 向待检细菌标本或细胞标本中加入培养液， 制作成液态检验标本， 所述的培养液包括有营养物质、 胶体原料；
( 3 )液态检验标本中加入可与胶体原料反应形成凝胶状的助凝剂， 制成 凝胶检验标本；
(4 ) 在凝胶检验标本的表面上贴药物纸片；
( 5 ) 将其放入培养箱中培养；
( 6 ) 观测药物抑菌圈， 确定对药物的敏感性。
上述细菌、 细胞药敏检验方法， 所述培养液包括有细菌细胞生长指示剂。 上述细菌、细胞药敏检验方法， 将液态检验标本放入培养箱中增殖培养 2 ― 4天。
上述细菌、 细胞药敏检验方法， 所述培养液中包括有抑菌剂。
上述细菌、 细胞药敏检验方法， 所述培养液中的营养物质、 胶体原料之 间的配比为： 每 100ml营养物质中加胶体原料 0.8g -— 2g; 每 100ml营养物质 配助凝剂 O. lg -— 0.2g。
. 上述细菌、 细胞药敏检验方法， 所述培养液中的营养物质、 胶体原料、 细菌细胞生长指示剂之间的配比为： 每 100ml营养物质中加胶体原料
0.8g --- 2g;加细菌细胞生长指示剂 2mg—- 20mg;每 100ml营养物质配助凝剂
O. l g— - 0.2g。
上述细菌、 细胞药敏检验方法， 所述培养液中的营养物质、 胶体原料、 细菌细胞生长指示剂、 抑菌剂之间的配比为： 每 100ml营养物质中加胶体原 料 0.8g --- 2g; 加细菌细胞生长指示剂 2mg—- 20mg; 加抑菌剂适量； 每 100ml 营养物质配助凝剂 O.lg -— 0.2g o . 上述细菌、 细胞药敏检验方法， 其培养液按一次使用的数量分装并密封 保存。
上述细菌、 细胞药敏检验方法的药敏检验装置， 包括有周壁和底部封闭、 上端敞开结构形式的盒体， 在盒体内腔中放置有助凝剂。
上述药敏检验装置， 在盒体内腔的底部设有助凝层， 助凝层由缓释体和 附着在缓释体中的助凝剂组成。
述的药敏检验装置， 在盒体内腔的底部设有助凝层， 助凝层为由粘合剂 与助凝剂混合后涂在盒体底部的结构形式。
上述药敏检验装置， 在盒体的底部设有标尺。
上述药敏检验装置， 在盒体的敞开端设有与其配套的盒盖。
本发明细菌、 细胞药敏检验方法， 其培养液中的营养物质包括有营养组 份和缓冲剂， 作用是为细菌或细胞生长提供营养和控制培养液的 PH值。 对 不同的细菌或细胞， 可以选用不同的营养物质配方。 例如， 结核杆菌可以选 用 7H9配方， 普通细菌或真菌可以选用营养肉汤配方， 肿瘤细胞可以选用细 胞培养基配方。 培养液中的胶体原料的作用是在培养液中加入助凝剂前， 胶 体原料溶解在其中， 使培养液呈液态， 在培养液中加入助凝剂后， 胶体原料 与助凝剂反应， 使培养液在一定时间内形成稳定的凝胶， 可以选用 0.5-1 .5% 海藻酸钠或者 0.5-2.0%果胶、 0.5-1 .5%卡拉胶等。 培养液中的细菌细胞 生长指示剂的作用是细菌或细胞在生长繁殖过程中会分解 MTT形成蓝色点状 色团， 从而在短时间内每一个细菌或细胞形成一个蓝色的肉眼可见的颗 粒状点， 可以根据肉眼或低倍显微镜准确观察点的性质和数量来确定细 胞或细菌的生长状况， 可以选用 3-(4， 5-二甲基噻唑 -2)-2， 5-二苯基四 氮唑溴盐 （其商品名为噻唑蓝， 英文名
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) 。
在培养液中加抑菌剂主要用于结核分枝杆菌药敏试验， 其作用是抑制抗酸 杆菌以外的其它杂菌生长。
本发明细菌、 细胞药敏检验方法使用的助凝剂， 其作用是助凝剂与培养 液中的胶体原料反应， 使培养液在一定时间内形成稳定的凝胶。 可以选用钙 离子或镁离子， 硼砂， 枸橼酸钾等。
由于本发明细菌、 细胞药敏检验方法， 其药敏检验装置包括有周壁和底 部封闭、 上端敞开结构形式的盒体， 在盒体中放置有助凝剂， 在培养液中加 入有胶体原料， 液态检验标本是利用培养液制成的， 因此， 在液态检验标本 中有胶体原料， 液态检验标本在倒入药敏检验装置内以前是呈液态的， 在倒 入药敏检验装置后， 检验标本中的胶体原料与药敏检验装置中的助凝剂反应， 可以在常温下快速形成稳定的凝胶状， 形成凝胶检验标本， 这样， 药物纸片 可以贴在凝胶检验标本的表面上， 因此， 可以用药物纸片检验细菌或细胞对 药物的敏感性。
本发明与现有的细菌、 细胞药敏检验方法相比， 整个检验过程时间短的 原因是： ①充分利用了患者提供的标本中的待检细菌或细胞。 患者提供的标 本中的细菌或细胞绝大部分被浓集用于制作检验标本， 而检验标本又全部被 用于做药敏试验， 因此， 患者提供的标本中的待检细菌或细胞得到了充分的 利用。 ②可以省略掉增殖或分离培养培养阶段或减少增殖或分离培养培养时 间。 对一般的细菌或细胞进行药敏检验， 同时做 6个药物纸片试验就能满足 要求， 因此， 药敏检验装置内腔的面积可以设计为同时做 6个药物纸片的药 敏试验。 由于药敏检验是对细菌或细胞的耐药性进行检测， 在进行药敏检验 以前， 对患者都进行了确定诊断。 对已经确诊的一般患者而言， 由于本发明 细菌、 细胞药敏检验方法充分利用了其提供的标本中的细菌或细胞， 这样在 进行药敏试验时， 药敏检验装置内腔中单位面积的细菌或细胞数量较大， 足 以满足药敏试验对细菌或细胞的数量要求， 不再需要通过增殖培养增加细菌 或细胞数量。 对在确定诊断时确认患者提供的标本含待检细菌量较少， 则应 该将制作成的液态检验标本置于培养箱中进行增殖培养， 之后再将液态检验 标本倒入药 (检验装置中进行药敏试验。 由于本发明的增殖培养是在液态培 养基中进行， 相对而言， 细菌在液态培养基中的增殖速度比在固体培养基上 的增殖速度快， 再加上充分利用了患者提供的标本中的待检细， 使增殖培养 前的初始数量较大， 因此， 增殖培养的时间很短。 例如对结核杆菌， 按照中 国疾病预防中心编写的， 由中国协和医科大学出版社 2004年 7月 1 日出版的 《中国结核病防治规划 痰涂片镜检质量保证手册》 中规定的， 采用萋一尼. 氏染色法， 如果镜检结果为抗酸杆菌阳性 （4+) ： 10条 /每视野， 可以不 进行增殖培养而直接进入到药敏检验阶段， 这样， 本发明就省略掉了增殖培 养阶段， 因此， 节省了整个试验时间； 如果镜检结果为抗酸杆菌阳性 （3+) ： 1一 9条 /每视野及以下， 则应该进行增殖培养， 由于在液态培养基中增殖速 度快， 加上初绐菌量大， 相对现有的增殖培养所需的时间而言， 所需的时间 短， 因而节约了整个试验的时间； ③药敏检验阶段时间短。 由于起始数量大， 比较容易形成药物抑菌圈， 与现有的纸片检验法相比所需的时间短， 因而节 约了整个试验的时间。 再加上细菌细胞生长指示剂的显示作用， 能更好地显 示细菌或细胞的繁殖生长情况和药物抑菌圈， 更便于观察检测， 可以进一步 减少试验时间。 经发明人反复试验证明， 利用本发明细菌、 细胞药敏检验方 法进行结核杆菌药敏试验， 可以省略增殖培养阶段， 即使需要增殖培养， 也 只需 2— 4天， 药敏检验阶段只需 5— 7天， 增整个检验过程不到 14天。 综上 所述， 本发明实现了检验过程时间短的目的。
本发明细菌、 细胞药敏检验方法投资少、 使用成本低的原因是： 实施本 发明所需的设备与现有的纸片检验法基本相同， 只需配套离心装置、 药敏检 验装置等。 上述的离心装置、 药敏检验装置等制造简单， 生产成本低， 因此， 整套设备的投资少； 实施本发明需要配套使用一次性试验容器以及包括营养 物质、 缓冲剂、 胶体原料、 指示剂等试剂， 这些一次性试验容器和试剂的成 本低且使用的数量少， 加上本发明仪器设备的投资少使用成本低， 进一步减 少了本发明的使用成本。 因此， 本发明能有效地实现投资少、 使用成本低的 目的。
本发明细菌、 细胞药敏检验方法使用安全方便的原因是： 本发明浓集待 检标本、 结果伴读均可以在一次性密闭容器内进行， 不会污染其他器材， 操 作完成后进行统一消毒处理， 并且整个检验过程手工操作的程序很少且简单， 因此， 能最大程度的减少了细菌对工作人员和环境的影响， 大大提高了试验 的生物安全性。 本发明由于将一次使用的培养液灌装在密封瓶中， 使用时， 只需打开密封瓶， 将培养液与浓集的细菌或细胞混均， 之后将其倒入药敏检 验装置中即可， 操作简单方便， 因此， 本发明能有效地实现使用安全方便的 目的。
附图说明
下面结合附图和实施例对本发明细菌、 细胞药敏检验方法及药敏检验装 置作进一步说明。
图 1所示为本发明中的药敏检验装置的主视图剖视示意图。
图 2为图 1所示的仰视图示意图。 1、 盒体， 2、 助凝层， 3、 盒盖， 4、 标尺。
具体实施方式
如图所示， 盒体 （1 ) 为周壁和底部封闭、 上端敞开的结构形式， 在盒体 ( 1 ) 内设有助凝层 （2 ) ， 助凝层 （2 ) 由缓释体和附着在缓释体中的助凝剂 组成， 在盒体 （1 ) 的底部设有标尺 （4) ， 在盒体 （1 ) 的敞开端配套有盒盖
( 3 ) 。
本发明的药敏检验装置， 其助凝层 （2 ) 中的缓释体可以是缓释膜， 当将 培养液倒入盒体 （1 ) 中后， 该缓释膜可以溶解于培养液中， 助凝剂附着在缓 释体中； 也可以采用糖与助凝剂混合， 利用喷涂的方法在盒体 （1 ) 中形成助 凝层。
下面给出本发明细菌、 细胞药敏检验方法及药敏检验装置用于结核杆菌 药敏试验的实施例：
对采用萋一尼氏染色法进行确定诊断， 镜检结果为抗酸杆菌阳性 （4+) ： 10条 /每视野的进行药敏试验：
1、 将患者晨痰标本留取 5ml用 4%氢氧化钠消化；
2、 置于离心机中， 4500转 10分钟离心分离， 将标本中结核杆菌充分沉 淀；
. 3、 去掉上清液；
4、 用 20ml培养液将沉淀物洗脱制成液体菌悬液形式的液态检验标本；
5、 将液态检验标本均匀倒在药敏检验装置的盒体中， 培养液中的胶体原 料与盒体中的助凝剂反应， 在 3小时内凝固成稳定的凝胶， 形成凝胶检验标 7、 盖上盒盖， 将其置于 37°C培养箱中培养 5-7天， 结核杆菌在生长繁殖 过程中会分解 MTT形成蓝色点状色团， 如果药物对结核杆菌有抑制作用则贴 有药物纸片的凝胶检验标本周围不能形成蓝色团， 因而形成抑菌圈；
8、 根据抑菌圈大小判断药物对细菌的作用效果。
本实施例中的 20 ml培养液采用的配方为： 营养物质为 7H9配方 20ml、 细菌细胞生长指示剂为 3-(4， 5-二甲基噻唑 -2)-2， 5-二苯基四氮唑溴盐 0.4mg、 胶体原料为 0.5-1 .5%海藻酸钠 0. 17g、 抑制剂为 0. 4mg孔雀绿； 本 实施例中的助凝剂为钙离子 0.02g。
本实施例中的 20 ml培养液采用的配方还可以是： 营养物质为 7H9配方 20ml , 细菌细胞生长指示剂为 3-(4， 5-二甲基噻唑 -2)-2， 5-二苯基四氮唑 溴盐 0.5mg、 胶体原料为 0.5-1 .5%海藻酸钠 0. 3g、 抑制剂为 0. 4mg孔雀绿 0.8ml ; 本实施例中的助凝剂为钙离子 0.03g。
本实施例中的 30 ml培养液采用的配方还可以是： 营养物质为 7H9配方 20ml、 细菌细胞生长指示剂为 3-(4， 5-二甲基噻唑 -2)-2， 5-二苯基四氮唑 溴盐 4mg、胶体原料 0. 4g、抑制剂为 0. 4mg孔雀绿。本实施例中的助凝剂为 钙离子 0.04g。
对采用萋一尼氏染色法进行确定诊断， 镜检结果为抗酸杆菌阳性 （3+) ： 1-9条 I每视野及以下的进行药敏试验：
1、 将患者晨痰标本留取 5ml用 4%氢氧化钠消化；
2、 置于离心机中， 4500转 10分钟离心分离， 将标本中结核杆菌充分沉 淀；
3、 去掉上清液；
4、 用 20ml培养液将沉淀物洗脱制成液体菌悬液形式的液态检验标本； . 5、将液态检验标本置于 37°C培养箱中增菌培养 2-4天， 观察到液态检验 标本呈现淡蓝色改变；
6、 将增菌后的液态检验标本均匀倒在药敏检验装置的盒体中， 培养液中 的胶体原料与盒体中的助凝剂反应， 在 3小时内凝固成稳定的凝胶， 形成凝 胶检验标本；
7、 在凝胶检验标本表面设定的位置贴上欲测药物纸片；
8、 盖上盒盖， 将其置于 37°C培养箱中培养 5-7天， 结核杆菌在生长繁殖 过程中会分解 MTT形成蓝色点状色团， 如果药物对结核分枝杆菌有抑制作用， 则结核分枝杆菌不能正常繁殖， 在贴有药物纸片的凝胶检验标本周围不能形 成蓝色团， 因而形成抑菌圈；
9、 根据抑菌圈大小判断药物对细菌的作用效果。
本实施例中的 20 ml培养液采用的配方还可以是： 营养物质为 7H9配方 20ml , 细菌细胞生长指示剂为 3-(4， 5-二甲基噻唑 -2)-2， 5-二苯基四氮唑 •溴盐 0.5mg、胶体原料为 0.5-1 .5%海藻酸钠 0. 3g、抑制剂为 0. 4mg孔雀绿。 本实施例中的助凝剂为钙离子 0.03g。
下面给出本发明细菌、 细胞药敏检验方法及药敏检验装置用于普通细菌 或真菌药敏试验的实施例：
1、 将分离培养后的目标菌落用 20ml培养液制成液体菌悬液形式的液态 检验标本；
2、 将液态检验标本均匀倒在药敏检验装置的盒体中， 培养液中的胶体原 料与盒体中的助凝剂反应， 在 3小时内凝固成稳定的凝胶， 形成凝胶检验标 本；
3、 在凝胶检验标本表面设定的位置贴上欲测药物纸片； 4、 盖上盒盖， 将其置于 37°C培养箱中培养 12- 24h， 普通细菌或真菌在 生长繁殖过程中会分解 MTT形成蓝色点状色团， 如果药物对细菌有抑制作用， 则细菌不能正常繁殖， 在贴有药物纸片的凝胶检验标本周围不能形成色团， 因而形成抑菌圈；
5、 根据抑菌圈大小判断药物对细菌作用效果。
本实施例中的 20 ml培养液采用的配方为： 营养物质为营养肉汤配方 20ml、 细菌细胞生长指示剂为 3-(4， 5-二甲基噻唑 -2)-2， 5-二苯基四氮唑 溴盐 4mg、 胶体原料为 0.5-1.5%海藻酸钠 0. 4g; 本实施例中的助凝剂、为钙 离子 0.04g。
. 下面给出本细菌、 细胞药敏检验方法及药敏检验装置用于肿瘤细胞药敏 试验的实施例：
1、 将已经确诊的肿瘤患者手术切除物细胞进行分离和培养， 待一定数量 后置专用容器中进行保存备用。
2、 取 1ml含肿瘤细胞的上述溶液加入 20ml含有本发明的培养液中， 混 匀， 制成细胞悬液形式的液态检验标本；
3、 将液态检验标本均匀倒在药敏检验装置的盒体中， 培养液中的胶体原 料与盒体中放置的助凝剂结合， 在 3小时内凝固成稳定的凝胶， 形成凝胶检 验标本；
4、 在凝胶检验标本表面设定的位置贴上欲测药物纸片；
5、 盖上盒盖， 将其置于 37°C培养箱中培养 2-7天， 肿瘤细胞在生长繁殖 过程中会分解 MTT形成蓝色点状色团， 如果药物对肿瘤细胞有抑制作用， 则 细胞不能正常繁殖， 在贴有药物纸片的凝胶检验标本周围不能形成色团， 因 而形成抑菌圈； 6、 根据抑菌圈大小判断药物对肿瘤细胞作用效果。
本实施例中的 20 ml培养液采用的配方为： 营养物质为细胞培养基配方 20ml , 细菌细胞生长指示剂为 3-(4， 5-二甲基噻唑 -2)-2， 5-二苯基四氮唑 溴盐 5mg、胶体原料为 0.5-1 .5%海藻酸钠 0. 4mgo 本实施例中的助凝剂为钙 离子 0.04mg。

Claims

权利 要 求
1、 一种细菌、 细胞药敏检验方法， 包括：
( 1 ) 浓集待检细菌标本或细胞标本；
其特征是：
( 2 ) 向待检细菌标本或细胞标本中加入培养液， 制作成液态检验标本， 所述的培养液包括有营养物质、 胶体原料；
( 3 )液态检验标本中加入可与胶体原料反应形成凝胶状的助凝剂， 制成 凝胶检验标本；
(4) 在凝胶检验标本的表面上贴药物纸片；
( 5 ) 将其放入培养箱中培养；
( 6 ) 观测药物抑菌圈， 确定对药物的敏感性。
2、 根据权利要求 1所述的细菌、 细胞药敏检验方法， 其特征是： 所述培:. > 养液包括有细菌细胞生长指示剂。
3、 根据权利要求 1所述的细菌、 细胞药敏检验方法， 其特征是： 将液态 检验标本放入培养箱中增殖培养 2— 4天。
4、 根据权利要求 2所述的细菌、 细胞药敏检验方法， 其特征是： 将液态 检验标本放入培养箱中增殖培养 2— 4天。
5、 根据权利要求 1或 2或 3或 4所述的细菌、 细胞药敏检验方法， 其特 征是： 所述培养液中包括有抑菌剂。
6、 根据权利要求 1所述的细菌、 细胞药敏检验方法， 其特征是： 所述培 养液中的营养物质、 胶体原料之间的配比为： 每 100ml营养物质中加胶体原 料 0.8g -— 2g; 每 100ml营养物质配助凝剂 O. lg --- 0.2g。
7、根据权利要求 2或 3或 4所述的细菌、细胞药敏检验方法，其特征是: 所述培养液中的营养物质、 胶体原料、 细菌细胞生长指示剂之间的配比为: . - 每 100ml营养物质中加胶体原料 0.8g—- 2g; 加细菌细胞生长指示剂
2mg --- 20mg; 每 100ml营养物质配助凝剂 O.lg—- 0.2g。
8、 根据权利要求 5所述的细菌、 细胞药敏检验方法， 其特征是： 所述培 养液中的营养物质、 胶体原料、 细菌细胞生长指示剂、 抑菌剂之间的配比为： 每 100ml营养物质中加胶体原料 0.8g—- 2g; 加细菌细胞生长指示剂
2mg --- 20mg; 加抑菌剂适量； 每 100ml营养物质配助凝剂 O. lg --- 0.2g。
9、 根据权利要求 1或 2或 3或 4或 6所述的细菌、 细胞药敏检验方法， 其特征是： 培养液按一次使用的数量分装并密封保存。
10、 根据权利要求 5所述的细菌、 细胞药敏检验方法， 其特征是： 培养 液按一次使用的数量分装并密封保存。
1 1、 根据权利要求 7所述的细菌、 细胞药敏检验方法， 其特征是： 培养' 液按一次使用的数量分装并密封保存。
12、 根据权利要求 8所述的细菌、 细胞药敏检验方法， 其特征是： 培养 液按一次使用的数量分装并密封保存。
13、 一种用于权利要求 1所述细菌、 细胞药敏检验方法的药敏检验装置， 其特征是： 包括有周壁和底部封闭、 上端敞开结构形式的盒体 （1 ) ， 在盒体
( 1 ) 内腔中放置有助凝剂。
14、 根据权利要求 13所述的药敏检验装置， 其特征是： 在盒体 （1 ) 内 腔的底部设有助凝层 （2 ) ， 助凝层 （2 ) 由缓释体和附着在缓释体中的助凝 剂组成。
15、 根据权利要求 13所述的药敏检验装置， 其特征是： 在盒体 （1 ) 内 腔的底部设有助凝层 （2) ， 助凝层 （2) 为由粘合剂与助凝剂混合后涂在盒 体 （1) 底部的结构形式。
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